The Mechanism Of Nitric Oxide Regulated Low Temperature Tolerance Of Cucumber Seedlings

[Objective]Cucumber（Cucumis sativus L.）is one of the most economically valuable vegetable crops in the world.It is often attacked by low temperature in winter and spring cultivation.Therefore,the research on the mechanism of enhancing low temperature tolerance of cucumber has become an urgent problem to be solved.Nitric oxide（NO）,as a small signal molecule,participates in the regulation of plant growth and development,abiotic stress and other biological processes.However,the molecular mechanism of NO regulating plant stress resistance is still unclear.In addition,NO regulates plant abiotic stress tolerance is a complex and huge network.Although many studies have proved that NO interacts with phytohormone,reactive oxygen species（ROS）and other signal molecular to regulate plant stress tolerance.And the relationship between NO and glutathione（GSH）,as well as NO and melatonin（MT）in regulating cucumber low temperature tolerance needs further research.Therefore,the main purpose of this article is to expand the network of NO signal transduction regulating plant low temperature tolerance,and study the molecular mechanism of NO regulating plant low temperature tolerance as much as possible.[Methods]This study adopts the method of indoor artificial simulation of low temperature stress.We analyzed the chlorophyll fluorescence of cold sensitive cultivation’Jinyan No.4’and cold tolerant cultivation’Jinyou No.1’cucumber seedlings pretreated with exogenous sodium nitroprusside（SNP,NO donor）,and explored the effect of exogenous NO on the primary photochemical reaction and photosynthetic mechanism activity of photosystem II（PSII）under low temperature stress;The transcriptome analysis of’Jinyan No.4’and’Jinyou No.1’cucumber seedlings pretreated with exogenous NO was carried out to analyze the molecular mechanism of NO regulating the low temperature tolerance of cucumber seedlings;The antioxidant system,activity of mitochondrial electron transfer chain（ETC）complex,content of polyamines（PAs）,photosynthesis and chlorophyll fluorescence of cucumber seedlings pretreated with NO and GSH synthesis inhibitors（BSO）and NO cooperates with MT were analyzed to elucidate the role of interaction between NO and GSH,as well as NO and MT in regulating low temperature tolerance of cucumber seedlings.[Results]The main results of this paper are as follows:（1）Compared with’Jinyan No.4’,low temperature has less inhibition on the degree of damage to the oxygen evolution complex（OEC）,reduction rate of secondary electron acceptor（Q_A）and the electron transfer efficiency of’Jinyou No.1’.However,exogenous NO significantly improved the PSII photosynthetic efficiency and photosynthetic electron transfer rate,activated the PSII reaction center,effectively protected the OEC,improved the electron transfer rate of secondary electron acceptor Q_A to Q_B and the ability of plastid quinone（PQ）to accept electrons,maked Q_A quickly enter the oxidation state,promoted the smooth electron transfer,and reduced the energy capture of cucumber,thus protecting the PSII of cucumber,improving the low temperature tolerance of the two cultivated cucumber seedlings under low temperature stress.In addition,the effect of exogenous NO on’Jinyou No.1’cucumber seedlings is more significant under low temperature stress.（2）Transcriptomics analysis showed that exogenous NO could improve the low temperature tolerance of cucumber seedlings mainly by regulating phytohormone synthesis and signal transduction,and the regulatory effect of NO on’Jinyou No.1’was more significant than’Jinyan No.4’,which was mainly manifested in ethylene and jasmonic acid（JA）signal transduction and gibberellin（GA）biosynthesis.In addition,further analysis on the transcriptomics of’Jinyan No.4’found that exogenous NO regulated the transcription level of downstream target genes（PAL,MCM5,MCM6,GA3ox,EIN3,NPR1,Peroxidase,CSH,LHCA1,LHCB1,LHCB3,LHCB5）by regulating the expression of some key transcription factors（TFs）（MYB63,WRKY21,HD-ZIP and b-ZIP）under low temperature stress,thereby regulating photosynthesis,lignin synthesis,phytohormone signal transduction,phenylalanine metabolism,cell cycle and GA synthesis to regulate the low temperature tolerance of cucumber seedlings.（3）Under low temperature stress,BSO treatment can inhibit the activity of nitric oxide synthase-like（NOS-like）to a certain extent,indicating a relationship between NO and GSH in regulating cucumber low temperature tolerance.GSH participated in exogenous NO to increase the activity of antioxidant enzymes,reduce the accumulation of hydrogen peroxide（H₂O₂）,inhibit lipid peroxidation,increase the content of GSH and PAs,and maintain a higher GSH/GSSG redox state and activity of nitrosoglutathione reductase（GSNOR）to provide sufficient NO and GSH,and activate the mitochondrial electron transport chain complex activity（CI,CII,CIII,CIV）and ATP synthase activity（CV）to improve the low temperature tolerance of cucumber seedlings.（4）100μmol·L^-1 MT（MT 100）can effectively protect cucumber leaves from low temperature damage to PSII and increase the number of active reaction centers of PSII.In addition,both NO and MT treatment alone or NO combined with MT treatment can reduce the content of H₂O₂ and superoxide anion(O₂^·-),and increase the activities of superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）,ascorbic acid peroxidase（APX）,glutathione reductase（GR）,monodehydroascorbic acid reductase（MDHAR）and dehydroascorbic acid reductase（DHAR）,while the MT synthesis inhibitor（p-PCA）significantly inhibited the positive effects of exogenous NO on cucumber antioxidant system,indicating that MT acts upstream of NO and participates in the regulation of cucumber antioxidant system;NO and MT also participated in the regulation of photosynthetic system in cucumber seedlings under low temperature stress.Specifically,both exogenous NO and MT alone or NO+MT treatment can increase the maximum photochemical efficiency（Fv/Fm）,net photosynthetic rate（Pn）,actual photochemical efficiency[Y（II）],quantum yield[Y（NPQ）],photochemical quenching coefficient（q P）,non-photochemical quenching coefficient（q N）of PSII,while decrease the optical compensation point（I_c）and the quantum yield of non-regulated energy dissipation[Y（NO）].However,p-PCA and PTIO can inhibit the positive effects of NO and MT.[Conclusion]Exogenous NO can jointly regulate the low-temperature tolerance of cucumber seedlings at both physiological and molecular levels:（1）Exogenous NO enhances cucumber low-temperature tolerance by protecting the OEC,activating the active reaction center of PSII,reducing the absorption of light energy and promoting the transmission of photosynthetic electrons to regulate low temperature tolerance of cucumber,and exogenous NO has a more significant regulatory effect on’Jinyou No.1’.（2）Exogenous NO regulates the expression of target genes related to photosynthesis,lignin synthesis,plant hormone signal transduction,phenylalanine metabolism,cell cycle and GA synthesis induced by TFs to regulate the low temperature tolerance of cucumber.（3）GSH participates in NO scavenging excess ROS accumulation caused by low temperature,increasing polyamines（PAs）content,and increasing the activities of mitochondrial complex and adenosine triphosphate（ATP）synthase to endow cucumber with low temperature tolerance.（4）MT may act on the upstream of NO to improve the antioxidant capacity of cucumbers by directly scavenging reactive oxygen species or increasing the activity of antioxidant enzymes.However,there is no obvious upstream and downstream relationship between NO and MT in regulating photosynthesis.No matter in the regulation of antioxidant system or photosynthetic system,NO and MT show obvious synergistic effect.